Saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study

Objectives

Automated point-of-care molecular assays have greatly shortened the turnaround time of respiratory virus testing. One of the major bottlenecks now lies at the specimen collection step, especially in a busy clinical setting. Saliva is a convenient specimen type that can be provided easily by adult patients. This study assessed the diagnostic validity, specimen collection time and cost associated with the use of saliva.

Comparative Evaluation of the Diagenode Multiplex PCR Assay on the BD Max System versus a Routine In-House Assay for Detection of Bordetella pertussis

This study looked at 128 nasopharyngeal aspirates (NPA) and 162 throat swabs (TS) tested with the Diagenode multiplex assay on the BD Max system versus our in-house Bordetella pertussis PCR. Sensitivity and specificity were 97.3% and 100% for NPA and 88.3% and 98% for TS, respectively. Of positive NPA, 42.1% were coinfected with respiratory viruses.     

Comparison of Midturbinate Flocked-Swab Specimens with Nasopharyngeal Aspirates for Detection of Respiratory Viruses in Children by the Direct Fluorescent Antibody Technique

Paired nasopharyngeal aspirate (NPA) and midturbinate flocked-swab specimens from 153 children with respiratory symptoms were examined by the direct fluorescent antibody (DFA) technique. Seventy-four infants (49%) had a viral infection documented by DFA. The flocked-swab specimens had 93% sensitivity and 96.7% agreement with the NPA specimens, with a kappa coefficient of 93.4% (95% confidence interval, 0.877, 0.991).The direct fluorescent antibody (DFA) technique revolutionized the rapid detection of respiratory viruses. Since its inception in 1968, it has been one of the mainstays in clinical virology laboratories throughout the world (4). The ability of DFA to detect respiratory viruses depends on many things, but it all begins with good specimen collection. The nasopharyngeal aspirate (NPA) has been considered the best specimen to detect respiratory viruses in infants (4). However, it is difficult to collect because it requires special equipment, such as a catheter, trap, and vacuum source, and specialized training. A traditional nasopharyngeal swab is the next best specimen, especially in older children or adults, because it utilizes common supplies; however, the collection end of the swab, comprised of wound Dacron fibers, has limited absorbent capacity to trap virus-infected exfoliated epithelial cells. A nylon nasopharyngeal flocked swab with enhanced absorptive properties introduced in 2006 compared favorably to the NPA for the detection of respiratory viruses by DFA (2). Recently, a midturbinate flocked swab developed by Smieja, et al. (7), and marketed by Copan, Inc., has offered a more intuitive approach for the collection of nasopharyngeal specimens (1). It has compared favorably to the NPA and the flocked nasopharyngeal swab in the diagnosis of respiratory viruses by culture, antigen detection, and PCR, none of which require intact exfoliated epithelial cells for visualization; there is no published experience of midturbinate flocked swabs with DFA in children (1, 5, 6). The midturbinate flocked swab differs from the nasopharyngeal swab. It has a sampling depth indication gauge and also has a larger absorptive capacity than the smaller nasopharyngeal swab.The present study was designed to compare the efficacy of the midturbinate flocked swab with the NPA in the detection of respiratory viruses by DFA.The study was conducted from 5 January 2010 through 11 March 2010. All children 2 years of age or less admitted to the infant''s floor of the hospital with respiratory symptoms were enrolled. The study was reviewed by the Children and Youth Institutional Review Board, who waived the need for a formal review because the study was deemed an evaluation comparing a new specimen collection device to the standard nasopharyngeal aspirate; parents were allowed to opt out of the use of the new specimen device. A nasopharyngeal aspirate specimen was collected through one nostril. A second specimen was collected through the other nostril with a midturbinate FLOQ swab (Copan Diagnostics, Inc., Murrieta, CA) designed for children 2 years of age or less; the swab was inserted up to the collar on the shaft. Both specimens were placed in 3 ml of Copan UTM transport medium, transported to the virus laboratory, and processed within 6 h. The suspension was centrifuged, and the cellular pellet washed. The cells were then spotted to glass slides. The cells were stained for DFA using a D3 Ultra respiratory screening identification kit (Diagnostic Hybrids, Inc. [DHI], Athens, OH). The kit screened for respiratory syncytial virus (RSV), influenza viruses (IFV) A and B, parainfluenza viruses (PFV) 1, 2, and 3, and adenovirus (AdV). An additional stain for human metapneumovirus (hMPV) (DHI) was included. The DFA readers were not blinded to the specimen source. The degree of DFA agreement between specimens collected by NPA and midturbinate flocked swabs was calculated with Cohen''s kappa coefficient of agreement.One hundred fifty-three infants entered the study. Paired specimens were collected from every infant. Respiratory viruses were identified in 74 (48.6%). Respiratory syncytial virus was most frequent, found in 47 patients (30%), with hMPV in 25 (16.3%), PFV in 1 (0.7%), AdV in 1 (0.7%), and IFV in none (0.0%). The 2009 H1N1 influenza A virus had last been identified in the laboratory in November 2009, more than 1 month before the start of the study. DFA of NPA specimens identified all the viruses. DFA of the flocked-swab specimens failed to detect 4 RSV and 1 hMPV isolate that had been detected in the NPA specimens. The negative DFA test results on flocked-swab specimens agreed with the negative DFA test results on NPA specimens. Overall, the positive and negative DFA test results on flocked-swab specimens had 96.7% agreement with the DFA test results on NPA specimens, with a Kappa coefficient of 93.4% (95% confidence interval [CI], 0.877, 0.991; P < 0.00001). The sensitivity of the flocked swab was 93.2% (95% CI, 0.849, 0.978).The midturbinate flocked swab proved to be comparable to the NPA for the detection of common respiratory viruses, such as RSV and hMPV, in a DFA test in the present study. The absence of IFV and the low numbers of AdV and PFV isolates in specimens prevented an assessment of the swab''s utility in detecting these viruses; however, earlier studies with nasopharyngeal flocked swabs suggested that the midturbinate swab would give similar results (3). In an earlier study, the sensitivity of the NPA in detecting either IFV or RSV was greater than the sensitivity of flocked nasopharyngeal swabs, although the difference was not statistically significant; the differences may be attributed to the greater number of respiratory epithelial cells available for examination in NPA specimens (2). The advantage of the midturbinate collection over nasopharyngeal collection resides in the relative ease of collection and the resultant patient cooperation, especially among the very young; however, the observations made in the present study may not extend beyond the pediatric population.     

Neuraminidase-inhibitor resistance testing for pandemic influenza A (H1N1) 2009 in Ontario,Canada

Background

WNV epidemics occur worldwide, new WNV isolates were isolated in southern-east Europe belonging to WNV lineage 2. A first international proficiency study on WNV indicted that some laboratories were not able to detect WNV lineage 2 virus genome by their PCR diagnostic assays. Therefore an actual External Quality Assessment with both virus lineages was performed to monitor the improvements in molecular diagnostics.

Objectives

To asses the proficiency of laboratories to detect West Nile virus with molecular diagnostic tests.

Study design

A test panel of different WNV isolates and virus dilutions was given to 26 laboratories to test the samples with their routine diagnostic methods.

Results

Twenty-one participating laboratories provided 28 data set results. WNV lineage 1 was detected with high overall efficiency of 92% (67.9–100%) but two different WNV lineage 2 strains were detected at lower rates (mean = 73%, 67.9–75%) by the different PCR assays. 93% of the laboratories were able to detect a WNV lineage 1 with a concentration of 1.2 × 10⁴ copies/ml but the detection rate was decreased to 68% for 1.2 × 10³ copies/ml. One laboratory generated false-positive result from the non-virus control samples and 29% of the datasets showed false-positive results for non-WNV flavivirus samples.

Conclusions

The WNV EQA showed an improved proficiency of laboratories as compared to the first EQA. However, the data suggest that problems in the detection of both lineages were still present since the first proficiency test was performed in 2006. Further proceedings versus the detection of both lineages are needed particularly for in-house assays.     

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait

A metagenomic approach based on target independent next‐generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next‐generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non‐respiratory viruses were detected in 14% of the clinical samples, while sequences of non‐human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real‐Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections.

     

Spectrum of respiratory viruses circulating in eastern India: Prospective surveillance among patients with influenza‐like illness during 2010–2011

In developing countries, viruses causing respiratory disease are a major concern of public health. During January 2010–December 2011, 2,737 patients with acute respiratory infection from the outpatient departments as well as patients admitted to hospitals were screened for different respiratory viruses. Nasal and or throat swabs were collected and transported to the laboratory where initial screening of influenza A and influenza B viruses was performed. The samples were tested further for influenza C virus, parainfluenza viruses 1–4, human rhinovirus, metapneumovirus and respiratory syncytial virus by conventional RT‐ PCR. The study revealed that the majority of the patients were under 5 years of age; both due to their higher susceptibility to respiratory infections and presentation to hospitals. Out of 2,737 patients enrolled in this study, 59% were found positive for one or more respiratory viruses. Influenza B infection was detected in 12% of patients followed by influenza A (11.7%), respiratory syncytial virus (7.1%), parainfluenza virus‐2 (6%), metapneumovirus (3%), parainfluenza virus‐3 (1%), parainfluenza virus‐4 (0.6%), parainfluenza virus‐1 (0.3%), influenza C (0.2%) and human rhinovirus (0.2%). Distinct seasonal infection was observed only for influenza A and influenza B viruses. J. Med. Virol. 85:1459–1465, 2013 . © 2013 Wiley Periodicals, Inc.

     

Characterization of the nasopharyngeal viral microbiome from children with community‐acquired pneumonia but negative for Luminex xTAG respiratory viral panel assay detection

In the present study, 50 nasopharyngeal swabs from children with community‐acquired pneumonia (CAP) but negative for 18 common respiratory viruses, as measured by the Luminex xTAG Respiratory Viral Panel Assay, were subjected to multiplex metagenomic analyses using a next‐generation sequencing platform. Taxonomic analysis showed that all sequence reads could be assigned to a specific species. An average of 95.13% were assigned to the Bacteria kingdom, whereas, only 0.72% were potentially virus derived. This snapshot of the respiratory tract virome revealed most viral reads to be respiratory tract related, classified into four known virus families: Paramyxoviridae , Herpesviridae , Anelloviridae , and Polyomaviridae . Importantly, we detected a novel human parainfluenza virus 3 (HPIV 3) strain with a 32‐bp insertion in the haemagglutinin‐neuraminidase (HN) gene that produced a negative result in the Luminex assay, highlighting the strength of virome metagenomic analysis to identify not only novel viruses but also viruses likely to be missed by ordinary clinical tests. Thus, virome metagenomic analysis could become a viable clinical diagnostic method.

     

Comparison of Luminex xTAG® RVP fast assay and real time RT‐PCR for the detection of respiratory viruses in adults with community‐acquired pneumonia

Community‐acquired pneumonia (CAP) is the third cause of death worldwide. Viruses are frequently detected in adult CAP. Highly sensitive diagnostic techniques should be used due to poor viral shedding. Different sampling methods can affect viral detection, being necessary to establish the optimal type of sample for identifying respiratory viruses in adults. The detection rates of respiratory viruses by Luminex xTAG® RVP fast assay, real time RT‐PCR (rtRT‐PCR) (Sacace®), and immunofluorescence assay (IFA) in adult CAP were performed in nasopharyngeal swabs (NPS) and aspirates (NPA) from 179 hospitalized adults. Positivity was 47.5% for Luminex®, 42.5% for rtRT‐PCR (P = 0.3), and 2.7% for IFA (2.7%) (P < 0.0). The sensitivity, specificity, and kappa coefficient of xTAG® RVP compared with rtRT‐PCR were 84.2%, 79.6%, and 0.62%, respectively. Luminex® and rtRT‐PCR detected 65 (58.0%) and 57 (50.9%) viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P < 0.01). xTAG® RVP is appropriate for detecting respiratory viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs. J. Med. Virol. 88:1173–1179, 2016 . © 2016 Wiley Periodicals, Inc.

     

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.     

Comparison of the Eragen Multi-Code Respiratory Virus Panel with Conventional Viral Testing and Real-Time Multiplex PCR Assays for Detection of Respiratory Viruses

High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing.The rapid and accurate detection of respiratory viruses is clinically important. Potential advantages of specific viral detection include obtaining prognostic information, limiting additional diagnostic testing, instituting appropriate infection control precautions, and limiting unnecessary antibiotic usage. The use of respiratory specimens for this purpose is challenging because of the broad range of pathogens that may be present. This task is becoming even more complicated with the recent discoveries of several new viruses in respiratory tract samples, including human metapneumovirus (MPV) (19), coronaviruses (CoVs) NL63 (4, 20) and HKU1 (23), human bocavirus (3), and polyomaviruses KI (2) and WU (5).Molecular methods offer the advantages of rapidity and the ability to detect viruses regardless of the growth requirements or the availability of reagents for rapid diagnostic testing. An exciting recent advance is the development of multiplex molecular assays that allow the simultaneous detection of multiple targets in the same reaction (12, 13, 15, 16). One such test is the xTAG Respiratory Virus Panel, produced by Luminex Molecular Diagnostics (Toronto, ON, Canada). This test, which uses the Luminex-100 or -200 flow cell instrument (12), has been cleared by the Food and Drug Administration for use in the United States for the simultaneous detection and identification of multiple respiratory viruses from nasopharyngeal swab specimens from individuals with suspected respiratory tract infection. In the present study, we have evaluated a different respiratory multiplex PCR test, the PLx MultiCode Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI). Two important characteristics of the evaluation are, first, that we have included substantial numbers of specimens containing each of the common respiratory viruses detected by the test and, second, that we have designed and validated PCR assays to detect each of the respiratory viruses and used these assays to resolve discrepancies between the results of conventional testing (fluorescent-antibody [FA] staining and culture) and PLx-RVP.     

Cost Analysis of Multiplex PCR Testing for Diagnosing Respiratory Virus Infections

We performed a cost analysis study using decision tree modeling to determine whether the use of multiplex PCR testing for respiratory viruses (xTAG RVP test) is a more or less costly strategy than the status quo testing methods used for the diagnosis of respiratory virus infections in pediatric patients. The decision tree model was constructed by using four testing strategies for respiratory virus detection, viz., direct fluorescent-antibody staining (DFA) alone, DFA plus shell vial culture (SVC), the xTAG RVP test alone, or DFA plus the xTAG RVP test. A review of the charts of 661 pediatric patients was used to determine the length of hospital stay, the number of days in isolation, antibiotic usage, and all other medical procedures performed. The cost of hospitalization by diagnostic status was determined on the basis of the average cost per patient and the number of patients in each arm of the decision tree. The cost per case was the highest for DFA plus SVC at $3,914 (in Canadian dollars), and the lowest was for the xTAG RVP test alone at $3,623, while the costs of DFA alone ($3,911) and DFA plus RVP ($3,849) were intermediate. When all four diagnostic strategies were compared, the least costly strategy was the xTAG RVP test alone when the prevalence of infection was 11% or higher and DFA alone when the prevalence was under 11%. These data indicate a savings of $291 per case investigated if the strategy of using the xTAG RVP test alone was used to replace the status quo test of DFA plus SVC, resulting in a savings of $529,620 per year in direct costs for the four Hamilton, Ontario, Canada, hospitals on the basis of the testing of specimens from 1,820 pediatric inpatients. We conclude that the use of the xTAG RVP test is the least costly strategy for the diagnosis of respiratory virus infections in children and would generate a significant savings for hospitals.Clinical virology laboratories have historically used traditional methods, such as culture, direct fluorescent-antibody staining (DFA), and enzyme immunoassay, for the diagnosis of respiratory tract infections (3). DFA offers a rapid turnaround time for results but is labor-intensive and subjective and requires specific monoclonal antibodies and trained technologists. Both DFA and shell vial culture (SVC) are limited by the availability of monoclonal antibodies, precluding their use for the detection of newly discovered viruses. DFA has a low sensitivity for the detection of some viruses, especially adenovirus, and many laboratories reflex DFA-negative specimens into SVCs to improve the detection rates. For traditional methods such as DFA and SVC, turnaround times for results can be slow for laboratories handling large volumes of specimens. Rapid enzyme immunoassays have been used for the detection of influenza virus and respiratory syncytial virus (RSV), but these tests are only 50 to 70% sensitive (5, 15), which limits their use to specific point-of-care settings at times when the prevalence of infection is high.Over the past 10 years, nucleic acid amplification tests have been developed for an increasing number of respiratory viruses. Nucleic acid amplification tests, including PCR and nucleic acid-sequence based amplification, have shown enhanced sensitivity compared with the sensitivities of DFA and culture for the detection of a number of respiratory viruses (8). The emergence of five new respiratory viruses since 2000, including human metapneumovirus, the sudden acute respiratory syndrome-associated coronavirus, avian influenza virus H5N1, coronaviruses NL63 and HKU1, and human bocavirus, has presented new challenges for clinical laboratories. The absence of commercially available tests for the detection of these emerging viruses often leaves laboratories without the ability to diagnose these important virus infections. Multiplex PCR assays for the detection of multiple respiratory viruses have recently been introduced (for a review, see reference 8). These multiplex assays have heralded a new era in the molecular diagnostics of respiratory virus infections. Some of these tests are now commercially available and can detect up to 18 different respiratory viruses (6). A multiplex PCR test for respiratory viruses (the xTAG RVP test) is the first multiplex PCR to be cleared by the U.S. Food and Drug Administration and has been approved for use for the detection of 12 different respiratory viruses (16). The xTAG RVP test detects 30 to 40% more virus infections than DFA and culture, in part because it is more sensitive for the detection of traditional respiratory viruses, but it also detects nine additional viruses not detected by DFA and SVC (6, 7, 9, 10, 14). These newer multiplex tests are often costly, and the clinical and economic impacts of their implementation in routine hospital laboratories have not been evaluated. We therefore conducted a cost analysis study to compare the costs of the xTAG RVP test to those of conventional tests for the diagnosis of respiratory virus infections in hospitalized patients.     

Viruses associated with influenza‐like‐illnesses in Papua New Guinea, 2010

Influenza‐like‐illness can be caused by a wide range of respiratory viruses. The etiology of influenza‐like‐illness in developing countries such as Papua New Guinea is poorly understood. The etiological agents associated with influenza‐like‐illness were investigated retrospectively for 300 nasopharyngeal swabs received by the Papua New Guinea National Influenza Centre in 2010. Real‐time PCR/RT‐PCR methods were used for the detection of 13 respiratory viruses. Patients with influenza‐like‐illness were identified according to the World Health Organization case definition: sudden onset of fever (>38°C), with cough and/or sore throat, in the absence of other diagnoses. At least one viral respiratory pathogen was detected in 66.3% of the samples tested. Rhinoviruses (17.0%), influenza A (16.7%), and influenza B (12.7%) were the pathogens detected most frequently. Children <5 years of age presented with a significantly higher rate of at least one viral pathogen and a significantly higher rate of co‐infections with multiple viruses, when compared to all other patients >5 years of age. Influenza B, adenovirus, and respiratory syncytial virus were all detected at significantly higher rates in children <5 years of age. This study confirmed that multiple respiratory viruses are circulating and contributing to the presentation of influenza‐like‐illness in Papua New Guinea. J. Med. Virol. 86:899–904, 2014. © 2013 Wiley Periodicals, Inc.

     

Human parainfluenza virus types 1–4 in hospitalized children with acute lower respiratory infections in China

Human parainfluenza viruses (HPIVs) are an important cause of acute lower respiratory tract infections (ALRTIs). HPIV‐4, a newly identified virus, has been associated with severe ALRTIs recently. A total of 771 nasopharyngeal aspirate samples were collected from hospitalized children between March 2010 and February 2011. HPIVs were detected by Nest‐PCR, and other known respiratory viruses were detected by RT‐PCR and PCR. All amplification products were sequenced. HPIVs were detected in 151 (19.58%) patients, of whom 28 (3.63%) were positive for HPIV‐4, 12(1.55%) for HPIV‐1, 4 (0.51%) for HPIV‐2, and 107 (13.87%) for HPIV‐3. Only three were found to be co‐infected with different types of HPIVs. All HPIV‐positive children were under 5 years of age, with the majority being less than 1 year. Only the detection rate of HPIV‐3 had a significant statistical difference (χ² = 29.648, P = 0.000) between ages. HPIV‐3 and HPIV‐4 were detected during the summer. Sixty (39.74%) were co‐infected with other respiratory viruses, and human rhinovirus (HRV) was the most common co‐infecting virus. The most frequent clinical diagnosis was bronchopneumonia, and all patients had cough; some patients who were infected with HPIV‐3 and HPIV‐4 had polypnea and cyanosis. No significant difference was found in clinical manifestations between those who were infected with HPIV‐4 and HPIV‐3. Two genotypes for HPIV‐4 were prevalent, although HPIV‐4a dominated. HPIV‐4 is an important virus for children hospitalized with ALRTIs in China. HRV was the most common co‐infecting virus. Two genotypes for HPIV‐4 are prevalent, HPIV‐4a dominated. J. Med. Virol. 88:2085–2091, 2016. © 2016 Wiley Periodicals, Inc.

     

Respiratory viral infections are underdiagnosed in patients with suspected sepsis

The study aim was to investigate the prevalence and clinical relevance of viral findings by multiplex PCR from the nasopharynx of clinically septic patients during a winter season. During 11 weeks of the influenza epidemic period in January–March 2012, consecutive adult patients suspected to be septic (n = 432) were analyzed with cultures from blood and nasopharynx plus multiplex PCR for respiratory viruses on the nasopharyngeal specimen. The results were compared with those from microbiology analyses ordered as part of standard care. During the winter season, viral respiratory pathogens, mainly influenza A virus, human metapneumovirus, coronavirus, and respiratory syncytial virus were clinically underdiagnosed in 70% of patients positive by the multiplex PCR assay. During the first four weeks of the influenza epidemic, few tests for influenza were ordered by clinicians, indicating low awareness that the epidemic had started. Nasopharyngeal findings of Streptococcus pneumoniae and Haemophilus influenzae by culture correlated to pneumonia diagnosis, and in those patients laboratory signs of viral co-infections were common but rarely suspected by clinicians. The role of respiratory viral infections in patients presenting with a clinical picture of sepsis is underestimated. Specific antiviral treatment might be beneficial in some cases and may reduce spread in a hospital setting. Diagnosing viral infections may promote reduction of unnecessary antibiotic use. It can also be a tool for decisions concerning patient logistics, in order to minimize exposure of susceptible patients and personnel.     

Comparison of the Luminex Respiratory Virus Panel Fast Assay with In-House Real-Time PCR for Respiratory Viral Infection Diagnosis

The Luminex xTAG Respiratory Virus Panel (RVP) assay has been shown to offer improved diagnostic sensitivity over traditional viral culture methods and to have a sensitivity comparable to those of individual real-time nucleic acid tests for respiratory viruses. The objective of this retrospective study was to test a new, streamlined version of this assay, the RVP Fast assay, which requires considerably less run time and operator involvement. The study compared the performance of the RVP Fast assay with those of viral culture, a direct fluorescent assay (DFA), and a panel of single and multiplex real-time PCRs in the testing of 286 respiratory specimens submitted to the Edinburgh Specialist Virology Centre for routine diagnosis of viral infection between December 2007 and February 2009. At least one respiratory viral infection was detected in 13.6% of specimens by culture and DFA combined, in 49.7% by real-time PCR, and in 46.2% by the RVP Fast assay. The sensitivity and specificity of the RVP Fast assay compared to the results of real-time PCR as the gold standard were 78.8% and 99.6%, respectively. Real-time PCR-positive specimens missed by the RVP Fast assay generally had low viral loads or were positive for adenovirus. Additionally, a small number of specimens were positive by the RVP Fast assay but were not detected by real-time PCR. For some viral targets, only a small number of positive results were found in our sample set using either method; therefore, the sensitivity of detection of the RVP Fast assay for individual targets could be investigated further with a greater number of virus-positive specimens.Viral infections of the respiratory tract have traditionally been diagnosed in the laboratory by culture of respiratory specimens and direct fluorescent assay (DFA). However, the availability of real-time PCR has allowed us to detect respiratory viruses with greater sensitivity and shorter turnaround times (12). In recent years, a number of new respiratory viruses have been identified, so we must now consider a wider range of viruses in our diagnoses (see, e.g., references 1 and 14). However, the number of fluorophores that can be differentiated in a multiplex real-time PCR assay limits the number of viral targets that can be detected.One solution is to screen each specimen with several different multiplex real-time PCRs to cover a large number of viruses (4). An alternative, the xTAG respiratory virus panel (RVP) assay (Luminex Molecular Diagnostics Inc., Toronto, Canada), is based on suspension microarray technology, which enables the detection of a large number of targets in a single reaction (6, 9). The xTAG RVP assay has been shown to offer results comparable or superior to those of culture/DFA and nucleic acid tests for the diagnosis of respiratory viral infections (7, 10). Recently, the RVP assay has been used successfully for the detection of etiological agents in outbreaks of respiratory illness (3, 15).The latest version of this test, the RVP Fast assay, has a simpler protocol and a shorter turnaround time than the original assay but still detects 19 different viral and subtype targets: influenza A virus (with additional subtyping: H1, H3, and H5), influenza B virus, respiratory syncytial virus A (RSV-A), RSV-B, parainfluenza virus 1 (PIV-1), PIV-2, PIV-3, PIV-4, adenovirus, human metapneumovirus, coronaviruses 229E, NL63, OC43, and HKU1, enterovirus/rhinovirus (EV/RhV), and human bocavirus. Here we compare the performance of the RVP Fast assay with those of culture/DFA and in-house real-time PCR assays, using respiratory specimens collected for routine viral testing.